The long-term goal of this project is to determine the molecular and cellular mechanisms by which double stranded RNA-dependent protein kinase PKR controls viral growth and cellular homeostasis. PKR is an interferon (IFN)-regulated host defense enzyme essential for higher vertebrates to suppress the spread of different viruses. PKR is a key regulator of protein synthesis and gene transcription. Once activated, PKR phosphorylates the protein synthesis initiation factor, eIF2alpha regulating mRNA translation. In response to specific signals, PKR also mediates phosphorylation of important transcriptional regulators, including the inhibitor of NF-kappaB, IkappaB, and the transcription factors IRF-1 and ATF2. Thus PKR has emerged as a multifunctional protein, which regulates cell growth, differentiation, and apoptosis. The specific objectives of this proposal are to determine the mechanisms of activation of PKR by dsRNA, to define the role of PKR in regulating key cellular proteins, RNA Helicase A and B65alpha, and to investigate PKR activity in RNA interference. To address these objectives we propose the following specific aims: 1. To delineate mechanisms of viral or cellular dsRNA activation and inhibition of PKR, we will determine the biophysical nature of wild-type PKR in solution alone and in complexes with the PKR activator, HIV-1 TAR RNA using neutron scattering, multidimensional heteronuclear NMR spectroscopy and X-ray crystallography. 2. To define protein-protein and kinase-substrate interactions of PKR we will explore the interactions domains of PKR with RNA helicase A (RHA) and B56a, the regulatory subunit of PP2A. The PKR phosphorylation sites on RHA and B56a will be defined and we will test the hypothesis that the PKR regulation of RHA plays a role in the innate resistance to virus infection. 3. To investigate the role of PKR in mediating the activities of micro and short interfering RNAs we will study the interaction of PKR with the shared components of these pathways, define determinants for activation of the interferon system and investigate antiviral activities against different viruses.